p53 kills cells by target gene activation and transcription-independent action. However, a mechanism for the latter was unknown. In the previous grant period we discovered that a fraction of induced p53 rapidly trans-locates to the mitochondrial surface of cultured cells during p53-dependent death. Importantly, bypassing the nucleus by targeting p53 to mitochondria is sufficient to launch apoptosis, p53 translocation also occurs in vivo in irradiated thymocytes. We explored the apoptogenic mechanism of mitochondrial p53 and showed that the p53 protein itself directly induces permeabilization of the outer mitochondrial membrane by forming complexes with the protective BcIXL and Bcl2 proteins, resulting in rapid cytochrome C release. We modeled and mapped the interaction: p53 binds to BcIXL via its DNA binding domain. We probed the significance of mitochondrial p53 and showed that breast cancer-derived, transactivation-deficient missense mutants of p53 concomitantly loose the ability to interact with BcIXL. This opens the possibility that p53 mutations represent 'double-hits' by simultaneously abrogating the transcriptional and mitochondrial apoptotic activity of p53. We propose a pathway where p53 exerts a rapid and direct apoptogenic role at the mitochondria, thereby jump-starting and amplifying the transcription-based apoptotic action of p53. This proposal is now exploring the in vivo action of mitochondrial p53 by focusing on genetic experiments. Aim I further defines function and regulation of this pathway. Aim II uses retroviral gene transfer of mitochondrially-targeted wtp53 in the mouse Elx-myc lymphoma model to test for tumor killing apoptotic potential of pre-existing tumors. Aim llI generates a transgenic model to test mitochondrially-targeted wtp53 for tumor suppressive action during spontaneous tumorigenesis. If confirmed, the mitochondrial p53 pathway will open the door towards therapeutic exploitation since it could add an important synergistic modality to the p53-based arsenal of cancer therapeutics that are currently developed. [unreadable] [unreadable]